Transient receptor potential melastatin 3 ion channel (TRPM3) belongs to the TRP family of cation-permeable ion channels involved in many important biological functions such as pain transduction, thermosensation, and mechanoregulation. The channel was reported to play an important role in Ca(2+) homeostasis, but its gating mechanisms, functions, and regulation are still under research. Utilizing biophysical and biochemical methods, we characterized two independent domains, Ala-35-Lys-124 and His-291-Gly-382, on the TRPM3 N terminus, responsible for interactions with the Ca(2+)-binding proteins calmodulin (CaM) and S100A1. We identified several positively charged residues within these domains as having a crucial impact on CaM/S100A1 binding. The data also suggest that the interaction is calcium-dependent. We also performed competition assays, which suggested that CaM and S100A1 are able to compete for the same binding sites within the TRPM3 N terminus. This is the first time that such an interaction has been shown for TRP family members.

• MeSH
• kalmodulin chemie   genetika   metabolismus   MeSH
• kationtové kanály TRPM chemie   genetika   metabolismus   MeSH
• lidé MeSH
• missense mutace MeSH
• proteiny S100 chemie   genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kalmodulin MeSH
• kationtové kanály TRPM MeSH
• proteiny S100 MeSH
• S100A1 protein MeSH Prohlížeč
• TRPM3 protein, human MeSH Prohlížeč

TRPM3 has been reported to play an important role in Ca(2+) homeostasis, but its gating mechanisms and regulation via Ca(2+) are unknown. Ca(2+) binding proteins such as calmodulin (CaM) could be probable modulators of this ion channel. We have shown that this protein binds to two independent domains, A35-K124 and H291-G382 on the TRPM3 N-terminus, which contain conserved hydrophobic as well as positively charged residues in specific positions, and that these residues have a crucial impact on its binding. We also showed that the other Ca(2+) binding protein, S100A1, is able to bind to these regions and that CaM and S100A1 compete for these binding sites on the TRPM3 N-terminus. Moreover, our results suggest that another very important TRP channel activity modulator, PtdIns(4,5)P(2), interacts with the CaM/S100A1 binding sites on the TRPM3 N-terminus with high affinity.

• MeSH
• fluorescenční polarizace MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• kalmodulin metabolismus   MeSH
• kationtové kanály TRPM chemie   metabolismus   MeSH
• liposomy metabolismus   MeSH
• molekulární modely MeSH
• povrchová plasmonová rezonance MeSH
• proteiny S100 metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidylinositol-4,5-difosfát MeSH
• kalmodulin MeSH
• kationtové kanály TRPM MeSH
• liposomy MeSH
• proteiny S100 MeSH
• S100A1 protein MeSH Prohlížeč

Vagal afferents regulate numerous physiological functions including arterial blood pressure, heart rate, breathing, and nociception. Cell bodies of vagal afferents reside in the inferior vagal (nodose) ganglia and their stimulation by various means is being considered as a way to regulate cardiorespiratory responses and control pain sensations. Stimulation of the nodose by exposure to infrared light is recently being considered as a precise way to elicit responses. These responses would likely involve the activity of temperature-sensitive membrane-bound channels. While papers have been published to track the expression of these transient receptor potential ion channels (TRPs), further studies are warranted to determine the in situ expression of the endogenous TRP proteins in the nodose ganglia to fully understand their pattern of expression, subcellular locations, and functions in this animal model. TRP ion channels are a superfamily of Na+ /Ca2+ -channels whose members are temperature- and/or mechano-sensitive and therefore represent a potential set of proteins that will be activated directly or indirectly by infrared light. Here, we report the spatial localization of six TRP channels, TRPV1, TRPV4, TRPM3, TRPM8, TRPA1, and TRPC1, from nodose ganglia taken from juvenile male Sprague-Dawley rats. The channels were detected using immunohistology with fluorescent tags on cryosections and imaged using confocal microscopy. All six TRP channels were detected with different levels of intensity in neuronal cell bodies and some were also detected in axonal fibers and blood vessels. The TRP receptors differed in their prevalence, in their patterns of expression, and in subcellular expression/localization. More specifically, TRPV1, TRPV4, TRPA1, TRPM8, TRPC1, and TRPM3 were found in vagal afferent cell bodies with a wide range of immunostaining intensity from neuron to neuron. Immunostaining for TRPV1, TRPV4, and TRPA1 appeared as fine particles scattered throughout the cytoplasm of the cell body. Intense TRPV1 immunostaining was also evident in a subset of axonal fibers. TRPM8 and TRPC1 were expressed in courser particles suggesting different subcellular compartments than for TRPV1. The localization of TRPM3 differed markedly from the other TRP channels with an immunostaining pattern that was localized to the periphery of a subset of cell bodies, whereas a scattering or no immunostaining was detected within the bulk of the cytoplasm. TRPV4 and TRPC1 were also expressed on the walls of blood vessels. The finding that all six TRP channels (representing four subfamilies) were present in the nodose ganglia provides the basis for studies designed to understand the roles of these channels in sensory transmission within vagal afferent fibers and in the responses elicited by exposure of nodose ganglia to infrared light and other stimuli. Depending on the location and functionality of the TRP channels, they may regulate the flux of Na+ /Ca2+ -across the membranes of cell bodies and axons of sensory afferents, efferent (motor) fibers coursing through the ganglia, and in vascular smooth muscle.

• Klíčová slova
• TRPA1, TRPC1, TRPM3, TRPM8, TRPV1, TRPV4, autonomic nervous system, blood vessels, confocal microscopy, immunohistochemistry, nodose ganglion, vagus nerve,
• MeSH
• ganglion inferius metabolismus   MeSH
• kationtové kanály TRP * metabolismus   MeSH
• kationtové kanály TRPM * metabolismus   MeSH
• kationtové kanály TRPV MeSH
• krysa rodu Rattus MeSH
• nervus vagus metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kationtové kanály TRP * MeSH
• kationtové kanály TRPM * MeSH
• kationtové kanály TRPV MeSH
• TRPM3 protein, rat MeSH Prohlížeč
• Trpv4 protein, rat MeSH Prohlížeč

The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.

• Klíčová slova
• Transient Receptor Potential, cholesterol, lipid raft, methyl-beta-cyclodextrin, pain, sphingomyelinase,
• MeSH
• analgetika farmakologie   terapeutické užití   MeSH
• beta-cyklodextriny * farmakologie   MeSH
• bolest farmakoterapie   metabolismus   MeSH
• CHO buňky MeSH
• cholesterol metabolismus   MeSH
• Cricetulus * MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPM * metabolismus   genetika   MeSH
• lidé MeSH
• membránové mikrodomény metabolismus   účinky léků   MeSH
• modely nemocí na zvířatech * MeSH
• myši MeSH
• pregnenolon farmakologie   MeSH
• sfingomyelinfosfodiesterasa * metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• analgetika MeSH
• beta-cyklodextriny * MeSH
• cholesterol MeSH
• kationtové kanály TRPM * MeSH
• methyl-beta-cyclodextrin MeSH Prohlížeč
• pregnenolon MeSH
• pregnenolone sulfate MeSH Prohlížeč
• sfingomyelinfosfodiesterasa * MeSH
• TRPM3 protein, mouse MeSH Prohlížeč
• TRPM8 protein, mouse MeSH Prohlížeč

Transient receptor potential melastatin (TRPM) channels, a subfamily of the TRP superfamily, constitute a diverse group of ion channels involved in mediating crucial cellular processes like calcium homeostasis. These channels exhibit complex regulation, and one of the key regulatory mechanisms involves their interaction with calmodulin (CaM), a cytosol ubiquitous calcium-binding protein. The association between TRPM channels and CaM relies on the presence of specific CaM-binding domains in the channel structure. Upon CaM binding, the channel undergoes direct and/or allosteric structural changes and triggers down- or up-stream signaling pathways. According to current knowledge, ion channel members TRPM2, TRPM3, TRPM4, and TRPM6 are directly modulated by CaM, resulting in their activation or inhibition. This review specifically focuses on the interplay between TRPM channels and CaM and summarizes the current known effects of CaM interactions and modulations on TRPM channels in cellular physiology.

• Klíčová slova
• TRPM channels, calcium homeostasis, calmodulin, calmodulin binding site, regulation,
• MeSH
• kalmodulin * metabolismus   MeSH
• kationtové kanály TRPM * metabolismus   MeSH
• proteiny vázající vápník metabolismus   MeSH
• vápník metabolismus   MeSH
• vápníková signalizace MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• kalmodulin * MeSH
• kationtové kanály TRPM * MeSH
• proteiny vázající vápník MeSH
• vápník MeSH

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca2+ concentration ([Ca2+]i) remain unknown. We analyzed mechanisms regulating resting [Ca2+]i in dissociated rat melanotrophs by Ca2+-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca2+ channels (VGCCs) as well as removal of extracellular Ca2+ resulted in a rapid and reversible decrease in [Ca2+]i, indicating constitutive Ca2+ influx through L-type VGCCs. Reduction of extracellular Na+ concentration (replacement with NMDG+) similarly decreased resting [Ca2+]i. When cells were champed at -80 mV, decrease in the extracellular Na+ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na+ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K+ concentration was increased from 5 mM to 50 mM in the presence of K+ channel blockers, Ba2+ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca2+]i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca2+]i in rat melanotrophs.

• Klíčová slova
• Ca(2+) homeostasis, Cation conductance, Patch-clamp, Ca(2+) imaging, ruthenium red, Rat melanotrophs, Voltage-gated calcium channels,
• MeSH
• kationtové kanály TRPV antagonisté a inhibitory   metabolismus   MeSH
• krysa rodu Rattus MeSH
• melanotropní buňky metabolismus   MeSH
• metoda terčíkového zámku MeSH
• potkani Wistar MeSH
• rutheniová červeň farmakologie   MeSH
• sodík metabolismus   MeSH
• vápník metabolismus   MeSH
• vápníkové kanály - typ L metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kationtové kanály TRPV MeSH
• rutheniová červeň MeSH
• sodík MeSH
• vápník MeSH
• vápníkové kanály - typ L MeSH